We reported last year that tyrosine phosphorylation of middle T antigen appeared to be regulated by a cellular protein kinase(s). We have searched for such cellular enzyme and found that the epidermal growth factor (EGF) enhances tyrosine phosphorylation of middle T antigen. Since it is known that EGF receptor is a tyrosine kinase and that tyrosine phosphorylation of middle T antigen is considered to be essential for the induction of cellular transformation, it appears that extracellular mitogenic growth factors regulate the activity of tyrosine phosphorylation of middle T antigen and that mitogenic growth factors may play an essential role in inducing the transformation phenotype. We have constructed recombinant plasmids which express middle T antigen in E. coli. One of them have the amino-terminal polypeptide with 23 amino acids of Lambda phage 0 protein which is joined to the 21st amino acid of middle T antigen. This fused protein is not associated with a tyrosine kinase activity and it does not serve as an efficient phosphate acceptor when it is mixed with authentic middle T antigen. The results suggest that middle T antigen, at least as a primary translation product, is not itself a kinase. The antibodies directed against a nonapeptide, which represents a functionally important part of middle T antigen, label actin containing microfilament bundles of untransformed cells and immunoprecipitate 130K cellular protein. Possible significance of this crossreaction is under investigation. During characterization of mouse embryonic cell lines with respect to their permissivity for polyomavirus growth, we found an additional developmental stage, distinguishable from embryonal carcinoma cells and trophoblast cells, and which still supports viral replication.